1. The Field of the Invention
The present invention relates to the transport of biologically active agents across biological barriers. More specifically, the present invention relates to methods and compositions that enhance transport of polyanionic macromolecules such as DNA, RNA, antisense oligonucleotides and their analogs across biological barriers.
2. Technical Background
Gene therapy and antisense technology have been highly promoted for their potential to treat or cure a number of diseases. Many viral diseases and genetic conditions can potentially be treated by gene therapy. A great number of genes that play a role in previously untreatable diseases such as cancer, autoimmune diseases, cystic fibrosis and the like have been discovered. With the discovery of the gene involved, researchers have determined that the disease can be treated by either blocking a gene that is being over-expressed or by providing a copy of a malfunctioning gene. Often these treatments require the administration of DNA, RNA, antisense oligonucleotides, and their analogs to achieve a desired intracellular effect.
These treatment strategies have been shown to block the expression of a gene or to produce a needed protein in cell culture. However, a major problem with these promising treatments, is adapting them for use in vivo. For a compound to be an effective pharmaceutical agent in vivo, the compound must be readily deliverable to the patient, not rapidly cleared from the body, have a tolerable level of toxicity, and be able to reach the site within the body where it is needed.
However, macromolecules such as DNA, RNA, antisense oligonucleotides, and their analogs share similar, significant pharmaceutical problems. While these compounds are generally not toxic, if there are admistered orally, they do not reach the desired site because they are digested and metabolized. Injection of these polyanionic macromolecules increases the length of time the molecules are in the body, but does not target the specific area of need. Moreover they are subject to rapid degradation within the blood stream and clearance from the body.
Because DNA, RNA, and oligonucleotides are polyanionic macromolecules they do not readily cross biological barriers. The transfer of these materials into living cells is the major impediment to their use as therapeutic agents. An effective gene and oligonucleotide delivery system will need to bind to an appropriate cell, be internalized by endocytosis, escape from the lysosome and ultimately transfer the intact free DNA or oligonucleotides to the nucleus or plasma. In another words, the success of gene therapy and antisense therapy is largely dependent on achieving a delivery of nucleic acids in sufficient quantities, to the correct target site of action, and for the desired time frame.
Many different strategies, including both viral and non-viral systems, have been attempted for the effective delivery of genes and oligonucleotides. Each of these strategies has had varying degrees of success. However, none of them are safe and efficient enough for clinical use. Toxicity, transfection efficiency, nucleic acid (NA) degradation and free NA release are challenging problems for all of the current non-viral gene delivery systems, including liposomes and cationic polymers.
A particular problem with non-viral delivery systems is the balance between the stability of the NA/carrier complex and the ability of the carrier to release the NA in the targeted cell. The NA/carrier complex must be stable enough to remain intact in the circulation system, but yet unstable enough to release the free NA at the target site.
One approach that has been used to allow entry of the polyanionic macromolecules to the cell cytoplasm is complexing the polyanionic macromolecule to a highly polycationic polymer such as PEI. PEI is a highly polycationic synthetic polymer. It has been used for years in common processes such as paper production, shampoo manufacturing, and water purification. Recently, PEI has become one of the most successful polycation carriers used in oligonucleotide and DNA delivery.
PEI has been shown to be a highly efficient carrier for delivering oligonucleotides and plasmids, both in vitro and in vivo. PEI is available in both linear and branched forms. Because of its high positive charge density, PEI spontaneously forms interpolyelectrolyte complex (Polyion complex) with nucleic acid as a result of cooperative electrostatic interaction between the ammonium groups of the PEI and the phosphate groups of the nucleic acid. The ability of PEI to transfect a wide variety of cells is well established. Compared to other polycationic carriers, PEI has proved to be much better in protecting against nucleic acid degradation and releasing the nucleic acid to the cytoplasm after endocytosis.
The transfection mechanism has been explored by different laboratories, but still is not quite clear. It is generally accepted that PEI transfection of cells begins with the entry of PEI via endocytosis. Then the complex or the PEI buffers the acidic pH of the lysosome, protecting the nucleic acid degradation and causing an osmotic swelling/rupture of the vesicles. The rupture of the vesicle releases the nucleic acid into the cytoplasm. The dissociation of free nucleic acid from the cationic polymer is generally assumed to be accelerated by the replacement of cellular polyanionic molecules. It is believed that protonation of the PEI leads to an expansion of the polymeric network due to the intramolecular charge repulsion.
However, PEI is not a perfect transfecting agent. For example, the PEI/NA complex usually produces serious aggregations in physiological buffers. Moreover, the complexes show limited stability in the presence of serum and are rapidly cleared out of the bloodstream following systemic administration. Moreover, PEI has been consistently observed to be toxic both in vitro and in vivo. These properties have significantly limited the biomedical applications of PEIs.
To partially overcome the toxic effects of the PEI and the aggregation problems of the PEI/NA complex in biological buffers, the polymer has been conjugated or grafted with both hydrophilic and hydrophobic groups. Grafting of the PEIs with PEG results in copolymers that can form relatively stable DNA complexes in aqueous buffers. However, transfection activity of these systems is much lower than that of unmodified PEI (25 kDa). Partially propionyl acylated liner PEI (50 kDa and 200 kDa) also shows less toxicity, but again this modification compromises the transfection activity. Conjugation of targeting groups, such as transferrin, mannose, and galactose, increased the transfection efficiencies toward targeted tissue, but still do not solve the intrinsic toxicity problems associated with high molecular PEIs, because high molecular PEIs have to be used as precursors in order to get efficient transfection activities. Small sized PEIs are much less toxic, but unfortunately low molecular weights PEIs (less than 2,000 Dalton) were found to produce no or very low transfection activities in various conditions.
In light of the foregoing, it would be an advancement in the art to provide a method of delivering polyanionic macromolecules to target cells. It would be an additional advancement to provide a carrier molecule that could efficiently transport the polyanionic macromolecules to across biological barriers. A further advancement would be achieved if the carrier molecule showed reduced toxicity as compared to presently available compounds. It would be a further advancement if the carrier/macromolecule complex were stable exhibited serum stability. It would be a further advancement if carrier/macromolecule complex could readily disassociate within the target cell. It would be a further advancement to provide a carrier molecule that could be targeted to a specific tissue or cell type.